Smoothened polypeptides and methods of use

ABSTRACT

Disclosed is an isolated or purified polypeptide or peptidomimetic comprising an amino acid sequence of a portion of a Smoothened (SMO) protein, wherein the portion comprises an amino acid sequence of any of the intracellular loops of the SMO protein, a functional fragment thereof, or a functional variant of either the portion or the functional fragment, wherein the functional fragment comprises at least 7 contiguous amino acids of the intracellular loops, and wherein the functional fragment or functional variant inhibits proliferation of a diseased cell, or a fatty acid derivative thereof. Related conjugates, nucleic acids, recombinant expression vectors, host cells, and pharmaceutical compositions are further provided. Methods of inhibiting proliferation of a diseased cell, treating or preventing cancer, treating a neoplasm or psoriasis, and inhibiting the expression of genes involved in the Hedgehog signaling pathway, thereby inhibiting the Hedgehog signaling pathway, are furthermore provided by the invention.

CROSS-REFERENCE TO A RELATED APPLICATION

This patent application claims the benefit of U.S. Provisional PatentApplication No. 60/855,422, filed Oct. 31, 2006, which is incorporatedby reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 53,510 Byte ASCII (Text) file named“701943 Sequence_ST25.TXT,” created on Oct. 24, 2007.

BACKGROUND OF THE INVENTION

Cancer is caused by dysregulations of signal transduction pathways. Onesuch pathway is the Hedgehog (HH) signal transduction pathway, whichinvolves the Patch (Ptch) and Smoothened (SMO) proteins. The HH pathwayis essential for embryonic cell growth (Beachy et al., Nature, 432:324-331 (2004)) and was found to be dysregulated in several cancers,including breast cancer (Katano et al., Cancer Lett., 227: 99-104(2005)), prostate cancer (Sanchez et al., Proc. Natl. Acad. Sci. U.S.A.,101: 12561-12566 (2004)), stomach cancer (Berman et al., Nature, 425:846-851 (2003)), colon cancer (Douard et al., Surgery, 139: 665-670(2006)), liver cancer (Sicklick et al., Carcinogenesis, 27: 748-757(2006)), melanoma (Pons et al., Clin. Transl. Oncol., 8: 466-474(2006)), basal cell carcinoma (Lam et al., Oncogene, 18, 833-836(1999)), and medulloblastoma (Berman et al., Science, 297, 1559-1561(2002) and Romer et al., Cancer Res., 65, 4975-4978 (2005)). There is adesire for inhibitors of the HH pathway for use in treatment of cancers.

BRIEF SUMMARY OF THE INVENTION

The invention provides an isolated or purified polypeptide or apeptidomimetic, as well as a fatty acid derivative thereof. Thepolypeptide or peptidomimetic comprises an amino acid sequencecorresponding to a portion of a SMO protein, wherein the portioncomprises an amino acid sequence of any of SEQ ID NOs: 2 to 4, eachsequence of which generally corresponds to an intracellular loop of theSMO protein. The polypeptide or peptidomimetic can be a functionalfragment of the portion, which functional fragment comprises at least 7contiguous amino acids of SEQ ID NO: 2, 3, or 4. The polypeptide orpeptidomimetic can be a functional variant of the portion or of thefunctional fragment. The inventive polypeptides and peptidomimetics(including fatty acid derivatives thereof, functional fragments andfunctional variants) inhibit the HH pathway and/or proliferation of adiseased cell.

The invention also provides conjugates comprising any of the inventivepolypeptides or peptidomimetics, or fatty acid derivatives thereof.Further provided are nucleic acids encoding the inventive polypeptides,as well as related recombinant expression vectors and host cells.Pharmaceutical compositions comprising any of the inventivepolypeptides, peptidomimetics, fatty acid derivatives, conjugates,nucleic acids, and recombinant expression vectors are furthermoreprovided by the invention.

The inventive pharmaceutical compositions are useful for inhibitingproliferation of a diseased cell, such that the invention moreoverprovides a method of inhibiting proliferation of a diseased cell. Themethod comprises contacting the diseased cell with an inventivepharmaceutical composition in an amount effective to inhibitproliferation of the diseased cell.

The invention provides other methods of use of the inventivepharmaceutical compositions, including a method of treating orpreventing cancer in a host, a method of treating psoriasis in a host, amethod of treating a neoplasm in a host, and a method of inhibitingexpression of a gene selected from the group consisting of Gli-1, Gli-2,Gli-3, Ptch, Shh, Smo, and NES in a diseased cell.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 depicts the % of viable MCF-7 breast cancer cells (relative to acontrol) upon a 48-hour treatment as a function of concentration ofSMOi1-1, SMOi2-1, and SMOi3-1 lipidated polypeptides, in accordance withan embodiment of the invention.

FIG. 2 depicts the viable cell mass of gastric adenocarcinoma cells upontreatment with 0, 5, or 10 μM (white, black, lined bars, respectively)of cyclopamine, SMOi3-1, or SMOi2-1.

FIG. 3 depicts the % of viable MCF-7 cells (relative to a control) upona 48-hour treatment with polypeptides comprising amino acid sequencesbased on the third intracellular loop of the SMO protein, in accordancewith an embodiment of the invention.

FIG. 4 depicts the % of viable SK-Mel2 cells upon a 48-hour treatmentwith SMOi2-8 or retroinverso analogues, SMOi2-16 and SMOi2-17, inaccordance with an embodiment of the invention.

FIG. 5 depicts the relative expression of genes of the HH pathway inDU145 cells upon a 48-hour treatment with cyclopamine, SMOi3-1, orSMOi2-1 polypeptides, in accordance with an embodiment of the invention.

FIG. 6 depicts the % of viable SK-Mel2 cells upon a 60-hour treatmentwith SMOi2-8 or peptidomimetics containing a 4-benzoylphenylalanine(BPA) residue in place of the Trp residue at position 5 of SMOi2-8, inaccordance with an embodiment of the invention.

FIG. 7 depicts the toxicity of the second intracellular loop derivatives(SMOi2-12 (circles) and SMOi2-17 (squares)) as determined by MTT assayin SK-Mel2 melanoma cells after 48 h of exposure to the peptidecompounds, in accordance with an embodiment of the invention.

FIG. 8 depicts the fluorescence emission intensity measured for probeswith increasing SMOi2-8/WMC-77 ratio, in accordance with an embodimentof the invention.

FIG. 9 depicts the growth inhibition of breast cancer, melanoma,heptaoma, and pancreatic cancer cells upon exposure to SMOi2-12(diamonds) or SMOi2-20 (squares), in accordance with an embodiment ofthe invention.

FIG. 10 depicts the circular dichroism spectrum of SMOi2-8 (diamonds)and SMOi2-16 (triangles) peptides.

DETAILED DESCRIPTION OF THE INVENTION

SMO proteins are transmembrane proteins which function in the Hedgehog(HH) signal transduction pathway (see, for instance, Huangfu andAnderson, Development 133: 3-14 (2006)), which, as discussed is relatedto several cancers, e.g., breast cancer, prostate cancer, stomachcancer, etc. These proteins comprise an extracellular domain, seventransmembrane domains, three intracellular loops, and an intracellulardomain. SMO proteins resemble a G-protein coupled receptor (GPCR) ingeneral topology but appear to signal differently from the GPCRs.Examples of SMO proteins include human SMO proteins (e.g., GenBankAccession No. NP_(—)005622 (SEQ ID NO: 1)), as well as orthologsthereof, such as mouse SMO proteins (e.g., GenBank Accession No.NP_(—)795970), rat SMO proteins (e.g., GenBank Accession No.NP_(—)036939), fruit fly SMO proteins (e.g., GenBank Accession No.NP_(—)523443), zebra fish SMO proteins (e.g., GenBank Accession No.NP_(—)571102), chicken SMO proteins, (e.g., GenBank Accession No.AAB84389), African clawed frog SMO proteins (e.g., GenBank Accession No.AAK15464).

The invention provides an isolated or purified polypeptide comprising anamino acid sequence corresponding to a portion of a SMO protein, whereinthe portion comprises an amino acid sequence of any of SEQ ID NOs: 2 to4, each of which is identical to or substantially identical to anintracellular loop of the SMO protein. For example, SEQ ID NO: 2contains one additional amino acid (Leu) at the N-terminus of the secondintracellular loop of SMO.

As used herein, the term “polypeptide” refers to a single chain of aminoacids connected by one or more peptide bonds. In this regard, the termencompasses peptides, oligopeptides, and polypeptides of any length,provided that there is at least one peptide bond. For purposes herein,the polypeptide of the invention comprises at least 6 peptide bonds,e.g., 10 or more peptide bonds.

The inventive polypeptides comprise an amino acid sequence of a portionof a SMO protein. That is to say that the polypeptides of the inventiondo not encompass any full-length, wild-type SMO proteins, e.g., SEQ IDNO: 1. In this respect, the inventive polypeptides comprise less thanabout 780 amino acids of a wild-type SMO protein. For example, theinventive polypeptides comprise less than about 500 amino acids of awild-type SMO protein. Most preferably, the inventive polypeptidescomprise less than about 75, e.g., about 50, amino acids of a wild-typeSMO protein. Also preferred is that the inventive polypeptides compriseabout 10 to about 12 amino acids (excluding any CPP, as discussedherein).

The portion of the SMO protein, in accordance with an embodiment of theinvention, comprises an amino acid sequence of any of SEQ ID NOs: 2 to4. In this regard, the inventive polypeptides can comprise, consistessentially of, or consist of an amino acid sequence of SEQ ID NO: 2, 3,or 4.

Alternatively, the portion of the SMO protein comprises an amino acidsequence of any of SEQ ID NOs: 5 to 8. In this regard, the inventivepolypeptides can comprise, consist essentially of, or consist of anamino acid sequence of SEQ ID NO: 5, 6, 7, or 8.

Included in the scope of the invention are functional fragments of theinventive polypeptides described herein. The term “functional fragment”when used in reference to an inventive polypeptide refers to any part orportion of the polypeptide of the invention, which part or portionretains the biological activity of the polypeptide of which it is a part(the parent polypeptide). The functional fragment can be any fragmentcomprising contiguous amino acids of the polypeptide of which it is apart, provided that the functional fragment inhibits proliferation of adiseased cell. Functional fragments encompass, for example, those partsof an inventive polypeptide that retain the ability to inhibitproliferation, or treat or prevent a disease (e.g., cancer, neoplasm,psoriasis), to a similar extent, the same extent, or to a higher extent,as the parent polypeptide. In reference to the parent polypeptide, thefunctional fragment can comprise, for instance, about 10%, 25%, 30%,50%, 68%, 80%, 90%, 95%, or more, of the parent polypeptide.

The functional fragment can comprise additional amino acids at the aminoor carboxy terminus, or at both termini, e.g., amino acids not found inthe amino acid sequence of the parent polypeptide. Desirably, theadditional amino acids do not interfere with the biological function ofthe functional fragment, e.g., inhibit proliferation, or treat orprevent a disease (e.g., cancer, neoplasm, psoriasis). More desirably,the additional amino acids enhance the biological activity, as comparedto the biological activity of the parent polypeptide.

In a preferred embodiment of the invention, the functional fragmentcomprises at least 5 contiguous amino acids of SEQ ID NO: 2, 3, or 4 andinhibits proliferation of a diseased cell. In a more preferredembodiment of the invention, the functional fragment comprises at least7 contiguous amino acids of SEQ ID NO: 2, 3, or 4. In a furtherpreferred embodiment of the invention, the functional fragment comprisesat least 4 contiguous amino acids of SEQ ID NO: 2, 3, or 4 and inhibitsproliferation of a diseased cell. The functional fragment of theinvention can, for example, comprise an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 5 to 8. For instance, thefunctional fragment can comprise an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 9 to 33. Also, the functionalfragment of the invention can consist essentially of or consist of anamino acid sequence of any of SEQ ID NOs: 9 to 33.

Further included in the scope of the invention are functional variantsof the inventive polypeptides, as well as functional variants of theinventive functional fragments described herein. The term “functionalvariant” as used herein refers to a polypeptide having substantial orsignificant sequence identity or similarity to a parent polypeptide orparent functional fragment, which functional variant retains thebiological activity of the polypeptide or functional fragment of whichit is a variant. Functional variants encompass, for example, thosevariants of the inventive polypeptide described herein (the parentpolypeptide) and those variants of the functional fragment describedherein (the parent functional fragment) that retain the ability toinhibit proliferation of a diseased cell to a similar extent, the sameextent, or to a higher extent, as the parent polypeptide or parentfunctional fragment. In reference to the parent polypeptide or parentfunctional fragment, the functional variant can, for instance, be atleast about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acidsequence to the parent polypeptide or parent functional fragment.

The functional variant can, for example, comprise the amino acidsequence of the parent polypeptide or parent functional fragment with atleast one conservative amino acid substitution. In this regard, thefunctional variant can comprise the amino acid sequence of the parentpolypeptide or parent functional fragment with two, three, four, five,or more conservative amino acid substitutions. Alternatively oradditionally, the functional variants can comprise the amino acidsequence of the parent polypeptide or parent functional fragment with atleast one non-conservative amino acid substitution. In this regard, thefunctional variant can comprise the amino acid sequence of the parentpolypeptide or parent functional fragment with two, three, four, five,or more non-conservative amino acid substitutions. In this case, it ispreferable for the non-conservative amino acid substitution to notinterfere with or inhibit the biological activity of the functionalvariant. Preferably, the non-conservative amino acid substitutionenhances the biological activity of the functional variant, such thatthe biological activity of the functional variant is increased ascompared to the parent polypeptide or parent functional fragment.

The functional variants preferably comprise one or more conservativeamino acid substitutions. Conservative amino acid substitutions areknown in the art, and include amino acid substitutions in which oneamino acid having certain physical and/or chemical properties isexchanged for another amino acid that has the same chemical or physicalproperties. For instance, the conservative amino acid substitution canbe an acidic amino acid substituted for another acidic amino acid (e.g.,Asp or Glu), an amino acid with a nonpolar side chain substituted foranother amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile,Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted foranother basic amino acid (Lys, Arg, etc.), an amino acid with a polarside chain substituted for another amino acid with a polar side chain(Asn, Cys, Gln, Ser, Thr, Tyr, etc.), an aromatic amino acid (Trp, Phe,Tyr, etc.) for another aromatic amino acid, etc.

Desirably, the functional variants of the invention comprise at least 4contiguous amino acids of SEQ ID NO: 2, 3, or 4, have at least 75%sequence identity (e.g., 80%, 85%, 90%, 95% sequence identity) to theparent polypeptide or parent functional fragment, and inhibitproliferation of a diseased cell.

For example, the functional variant can be a functional variant of anyof SEQ ID NOs: 17 to 21 and 23 to 33. In this regard, the functionalvariants can comprise the amino acid sequence of any of SEQ ID NOs: 38to 54 and 57 to 59, wherein Xaa is selected from a group consisting ofTyr, Phe, or BPA.

Also, for example, the functional variant can be a functional variant ofSMOi2-8 (SEQ ID NO: 23) comprising the amino acid sequence of SMOi2-8with one of the amino acids at any of positions 1-7,9,11, and 12 issubstituted with Ala.

Alternatively, the functional variant can comprise a retroinversoanalogue of any of the inventive polypeptides or functional fragmentsdescribed herein. The term “retroinverso analogue” refers to apolypeptide comprising a reversed amino acid sequence of a parentpolypeptide, such that the amino acid sequence of the retroinversoanalogue (when read from the N-terminus to the C-terminus) is the sameas the amino acid sequence of the parent polypeptide when read from theC-terminus to the N-terminus. Furthermore, with respect to aretroinverso analogue, each of the amino acids is the D isomer of theamino acid, as opposed to the L isomer. For example, the retroinversoanalogue of the tripeptide Val-Ala-Gly has an amino acid sequenceGly-Ala-Val, in which each amino acid is the D isomer. With respect tothe invention, the functional variant preferably comprises aretroinverso analogue of SEQ ID NO: 23, 26, or 33. In this regard, thefunctional variant comprises the amino acid sequence of any of SEQ IDNOs: 34 to 37.

The polypeptides of the invention (including functional fragments andfunctional variants) can be obtained by methods known in the art.Suitable methods of de novo synthesizing polypeptides are described inreferences, such as Chan et al., Fmoc Solid Phase Peptide Synthesis,Oxford University Press, Oxford, United Kingdom, 2005; Peptide andProtein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; EpitopeMapping, ed. Westwood et al., Oxford University Press, Oxford, UnitedKingdom, 2000; and U.S. Pat. No. 5,449,752. Also, polypeptides can berecombinantly produced using the nucleic acids described herein usingstandard recombinant methods. See, for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3^(rd) ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, NY, 1994. Further, some of the polypeptides of theinvention (including functional fragments and functional variantsthereof) can be isolated and/or purified from a source, such as a plant,a bacterium, an insect, a mammal, e.g., a rat, a human, etc. Methods ofisolation and purification are well-known in the art. Alternatively, thepolypeptides described herein (including functional fragments andfunctional variants thereof) can be synthesized or obtained commerciallyfrom companies such as Synpep (Dublin, Calif.), Peptide TechnologiesCorp. (Gaithersburg, Md.), and Multiple Peptide Systems (San Diego,Calif.). In this respect, the inventive polypeptides can be synthetic,recombinant, isolated, and/or purified.

Also provided by the invention are peptidomimetics of any of theinventive polypeptides (including functional fragments and functionalvariants) described herein. The term “peptidomimetic” as used hereinrefers to a compound which has essentially the same general structure ofa corresponding polypeptide with modifications that increase itsstability or biological function. A peptidomimetic includes, forexample, those compounds comprising the same amino acid sequence of acorresponding polypeptide with an altered backbone between two or moreof the amino acids. Additionally, the peptidomimetic can comprisesynthetic or non-naturally occurring amino acids in place ofnaturally-occurring amino acids.

In a preferred embodiment, the peptidomimetic is a peptoid. The term“peptoid” as used herein refers to a peptidomimetic in which thesidechains of each amino acid is appended to the nitrogen atom of theamino acid as opposed to the alpha carbon. For example, peptoids can beconsidered as N-substituted glycines which have repeating units of thegeneral structure of NRCH₂CO and which have the same or substantiallythe same amino acid sequence as the corresponding polypeptide.

In another preferred embodiment, the peptidomimetic comprises an alteredbackbone in which the bond between each amino acid is methylated. Inthis regard, the peptidomimetic can comprise a methylated peptidebackbone of the following structure:

The polypeptides (including functional fragments and functionalvariants) and peptidomimetics of the invention can be of any length,i.e., can comprise any number of amino acids, provided that thepolypeptide (or functional fragment or functional variant thereof) orpeptidomimetic retains their biological activity, e.g., the ability toinhibit proliferation of a diseased cell, treat or prevent disease(e.g., cancer, neoplasm, psoriasis) in a host, etc. For example, theinventive polypeptide or peptidomimetic can be 50 to 5000 amino acidslong, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 30, 40,50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900,1000 or more amino acids in length. Preferably, the polypeptides of theinvention are 5 to 50 amino acids in length.

The polypeptides (including functional fragments and functionalvariants) and peptidomimetics of the invention can comprise syntheticamino acids in place of one or more naturally-occurring amino acids.Such synthetic amino acids are known in the art, and include, forexample, aminocyclohexane carboxylic acid, norleucine, α-aminon-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-benzoylphenylalanine,4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine,β-phenylserine, β-hydroxyphenylalanine, phenylglycine,α-naphthylalanine, cyclohexylalanine, cyclohexylglycine,indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, aminomalonic acid, aminomalonic acid monoamide,N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine,ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptane carboxylic acid,α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid,α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

The polypeptides (including functional fragments and functionalvariants) and peptidomimetics of the invention can be lipidated (e.g.,fatty acidated), glycosylated, amidated, carboxylated, phosphorylated,esterified, N-acylated, cyclized via, e.g., a disulfide bridge, orconverted into an acid addition salt and/or optionally dimerized orpolymerized, or conjugated.

In this regard, the invention further provides lipidated derivatives ofany of the polypeptides (including functional fragments and functionalvariants) and peptidomimetics of the invention. Lipidated derivatives ofthe invention encompass any of the polypeptides and peptidomimeticsdescribed herein comprising a lipid molecule. As used herein, the term“lipid molecule” refers to any molecule comprising a hydrophobic moietywhich facilitates the entry of the polypeptide (including functionalfragments and functional variants) or peptidomimetic across the cellmembrane and into the cell. The lipid can be any lipid known in the art,such as, for example, a fatty acid, a farnesyl group (e.g., farnesyldiphosphate), a geranylgeranyl group (e.g., geranylgeranyl diphosphate),a phospholipid group, glycophosphatidylinositol, phosphatidylserine,phosphatidylethanolamine, sphingomyelin, phosphatidylcholine,cardiolipin, phosphatidylinositol, phosphatidic acid,lysophosphoglyceride, and a cholesterol group.

Preferably, the lipidated derivative is a fatty acid derivative in whichthe polypeptide or peptidomimetic described herein comprises a fattyacid molecule. The fatty acid molecule can be any C₈ to C₂₀ fatty acid.The fatty acid molecule can be, e.g., lauric acid, palmitic acid,myristic acid, stearic acid, oleic acid, linoleic acid, α-linoleic acid,linolenic acid, arachidonic acid, timnodonic acid, docosohexenoic acid,erucic acid, arachidic acid, or behenic acid. The fatty acid mayoptionally contain additional functional groups, e.g., one or more aminogroups on any of the carbon atoms. In a preferred embodiment, the fattyacid molecule is a C₈ to C₁₆ fatty acid, for example, a C₁₆ fatty acid.In a more preferred embodiment, the fatty acid is palmitate.

As is true with respect to the lipid molecule, the fatty acid moleculecan be attached to any suitable part of the inventive polypeptide(including functional fragment and functional variant) orpeptidomimetic. In a preferred embodiment of the invention, the fattyacid derivative of the inventive polypeptide (including functionalfragment and functional variant) or peptidomimetic comprises a fattyacid molecule at the amino (N-) terminus, the carboxyl (C-) terminus, orboth the N- and C-termini.

The fatty acid molecule can be attached to the inventive polypeptide(including functional fragment and functional variant) or peptidomimeticdirectly or through a linker. In an embodiment of the invention, whenthe fatty acid molecule is at the C-terminus of the inventivepolypeptide or peptidomimetic, the fatty acid molecule is attachedthrough an amino acid linker selected from the group consisting of Lys,Cys, homocysteine (homoCys), Orn, α,γ-diaminobutyric acid, andα,β-diaminopropionic acid. In a preferred embodiment of the invention,the fatty acid molecule is attached through a Lys. In a more preferredembodiment of the invention, the fatty acid molecule is attached throughthe epsilon carbon of Lys.

In another preferred embodiment, when the fatty acid molecule is at theC-terminus of the inventive polypeptide or peptidomimetic, the fattyacid molecule is modified e.g., to include an amino group such as in amodified molecule of Formula I or Formula H, wherein Formula I isNH₂(CH₂)_(n)COOH and Formula II is CH₃(CH₂)_(m)CH(NH₂)COOH, wherein eachof n and in is 1 to 24. In this regard, the fatty acid molecule isattached to the carboxyl group of the C-terminal amino acid of thepolypeptide or peptidomimetic. Preferably, n or m is 8 to 16. Morepreferably, n or m is 16.

Alternatively, the inventive polypeptides (including functionalfragments and functional variants) or peptidomimetics described hereincan comprise a cell-penetrating peptide (CPP). Such a CPP facilitatesthe entry of the inventive polypeptide or peptidomimetic across the cellmembrane and into the cell. CPPs are known in the art. See, for example,Deshayes et al., Cell. Mol. Life. Sci. 62: 1839-1849 (2005);El-Andaloussi et al., Curr. Pharm. Design 11: 3597-3611 (2005); and Mäeand Langel, Curr. Opin. Pharmacol. 6: 509-514 (2006)). The CPP can beany of those known in the art, e.g., Transportan, VP22, Pep1, and thelike. Preferably, the CPP comprises an amino acid sequence of SEQ ID NO:78 or 79, which corresponds to the amino acid sequence of penetratin andTat (48-60), respectively.

The polypeptides (including functional fragments and functionalvariants) and peptidomimetics, including fatty acid derivatives thereof,of the invention can be a monomer peptide, or can be a dimer or multimerpeptide. For example, the polypeptide can be a dimer of the followinggeneral structure:

wherein Sequence X is selected from the group consisting ofAc-LAKFSTHWAYTL (all-D) (SEQ ID NO: 85); Ac-AKFSTHWAYTL (All-D) (SEQ IDNO: 86); and Ac-KFSTHWAYTL (All-D) (SEQ ID NO: 87); wherein each ofLinker 1 and Linker 2 is optionally present and each independently isGly, beta-Ala, aminopropionic acid, gamma-aminobutyric acid,aminocaproic acid, or aminohexanoic acid; wherein n and m is between 0and 6; wherein Y is K, C, homoCys, Orn, diaminopropanoic acid (DPA),diaminobutyric acid (DBA); and wherein the fatty acid is a stearic,palmitic, myristic, lauric, capric or caprilic acid. In a preferredembodiment, Sequence X is SEQ ID NO: 87, n is 4, m is 0, each of Linker1 and Linker 2 is beta-Ala, and the fatty acid is palmitate. Methods ofmaking dimeric and multimeric polypeptides are known in the art. See,for example, Wrighton et al., Nature Biotechnology 15: 1261-1265 (1997).A preferred method of making a dimeric polypeptide also is set forthherein as Example 1.

When the polypeptides (including functional fragments and functionalvariants) and peptidomimetics, including fatty acid derivatives thereof,of the invention are in the form of a salt, preferably, the polypeptidesor peptidomimetcs are in the form of a pharmaceutically acceptable salt.Suitable pharmaceutically acceptable acid addition salts include thosederived from mineral acids, such as hydrochloric, hydrobromic,phosphoric, metaphosphoric, nitric, and sulphuric acids, and organicacids, such as tartaric, acetic, citric, malic, lactic, fumaric,benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, forexample, p-toluenesulphonic acid.

Further provided by the invention is a nucleic acid encoding any of theinventive polypeptides, including functional fragments and functionalvariants, described herein. As used herein, the term “nucleic acid”encompasses “polynucleotide,” “oligonucleotide,” and “nucleic acidmolecule,” and generally refers to a polymer of DNA or RNA, which can besingle-stranded or double-stranded, synthesized or obtained (e.g.,isolated and/or purified) from natural sources, which can containnatural, non-natural or altered nucleotides, and which can contain anatural, non-natural or altered internucleotide linkage, such as aphosphoroamidate linkage or a phosphorothioate linkage, instead of thephosphodiester found between the nucleotides of an unmodifiedoligonucleotide. It is generally preferred that the nucleic acid doesnot comprise any insertions, deletions, inversions, and/orsubstitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Sambrook et al., supra, and Ausubel et al., supra. For example,a nucleic acid can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed upon hybridization (e.g.,phosphorothioate derivatives and acridine substituted nucleotides).Examples of modified nucleotides that can be used to generate thenucleic acids include, but are not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N²-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The nucleic acid can comprise any nucleotide sequence which encodes anyof the inventive polypeptides, including functional fragments andfunctional variants. For example, the nucleic acid can comprise anucleotide sequence encoding any of SEQ ID NOs: 2 to 29, 32, 33, 38 to48, 53 to 56, 59 to 66, 70 to 72, 76, and 80. The nucleic acidalternatively can comprise a nucleotide sequence which is degenerate toany of these sequences or a combination of degenerate sequences. Theinvention also provides an isolated or purified nucleic acid comprisinga nucleotide sequence which is complementary to the nucleotide sequenceof any of the nucleic acids described herein or a nucleotide sequencewhich hybridizes under stringent conditions to the nucleotide sequenceof any of the nucleic acids described herein.

The nucleic acids of the invention can be incorporated into arecombinant expression vector. In this regard, the invention providesrecombinant expression vectors comprising any of the nucleic acids ofthe invention. For purposes herein, the term “recombinant expressionvector” means a genetically-modified oligonucleotide or polynucleotideconstruct that permits the expression of an mRNA, protein, polypeptide,or peptide by a host cell, when the construct comprises a nucleotidesequence encoding the mRNA, protein, polypeptide, or peptide, and thevector is contacted with the cell under conditions sufficient to havethe mRNA, protein, polypeptide, or peptide expressed within the cell.The vectors of the invention are not naturally-occurring as a whole.However, parts of the vectors can be naturally-occurring. The inventiverecombinant expression vectors can comprise any type of nucleotides,including, but not limited to DNA and RNA, which can be single-strandedor double-stranded, synthesized or obtained in part from naturalsources, and which can contain natural, non-natural or alterednucleotides. The recombinant expression vectors can comprisenaturally-occurring, non-naturally-occurring internucleotide linkages,or both types of linkages. Preferably, the non-naturally occurring oraltered nucleotides or internucleotide linkages does not hinder thetranscription or replication of the vector.

The recombinant expression vector of the invention can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The vector can be selected from the group consisting of thepUC series (Fermentas Life Sciences), the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10,λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used.Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, therecombinant expression vector is a viral vector, e.g., a retroviralvector.

The recombinant expression vectors of the invention can be preparedusing standard recombinant DNA techniques described in, for example,Sambrook et al., supra, and Ausubel et al., supra. Constructs ofexpression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColEl, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected hosts. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like. Suitable marker genes for the inventiveexpression vectors include, for instance, neomycin/G418 resistancegenes, hygromycin resistance genes, histidinol resistance genes,tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or normativepromoter operably linked to the nucleotide sequence encoding themodified TCR, polypeptide, or protein (including functional portions andfunctional variants thereof), or to the nucleotide sequence which iscomplementary to or which hybridizes to the nucleotide sequence encodingthe modified TCR, polypeptide, or protein. The selection of promoters,e.g., strong, weak, inducible, tissue-specific anddevelopmental-specific, is within the ordinary skill of the artisan.Similarly, the combining of a nucleotide sequence with a promoter isalso within the skill of the artisan. The promoter can be a non-viralpromoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, anSV40 promoter, an RSV promoter, and a promoter found in thelong-terminal repeat of the murine stem cell virus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression.

Further, the recombinant expression vectors can be made to include asuicide gene. As used herein, the term “suicide gene” refers to a genethat causes the cell expressing the suicide gene to die. The suicidegene can be a gene that confers sensitivity to an agent, e.g., a drug,upon the cell in which the gene is expressed, and causes the cell to diewhen the cell is contacted with or exposed to the agent. Suicide genesare known in the art (see, for example, Suicide Gene Therapy: Methodsand Reviews, Springer, Caroline J. (Cancer Research UK Centre for CancerTherapeutics at the Institute of Cancer Research, Sutton, Surrey, UK),Humana Press, 2004) and include, for example, the Herpes Simplex Virus(HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleosidephosphorylase, and nitroreductase.

The invention further provides a host cell comprising any of therecombinant expression vectors described herein. As used herein, theterm “host cell” refers to any type of cell that can contain theinventive recombinant expression vector. The host cell can be aeukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5α E. colicells, Chinese hamster ovarian cells, monkey VERO cells, COS cells,HEK293 cells, and the like. For purposes of amplifying or replicatingthe recombinant expression vector, the host cell is preferably aprokaryotic cell, e.g., a DH5α cell. For purposes of producing arecombinant modified TCR, polypeptide, or protein, the host cell ispreferably a mammalian cell. Most preferably, the host cell is a humancell. The host cell can be of any cell type, can originate from any typeof tissue, and can be of any developmental stage.

Also provided by the invention is a population of cells comprising atleast one host cell described herein. The population of cells can be aheterogeneous population comprising the host cell comprising any of therecombinant expression vectors described, in addition to at least oneother cell, e.g., a host cell (e.g., a T cell), which does not compriseany of the recombinant expression vectors, or a cell other than a Tcell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, ahepatocyte, an endothelial cell, an epithelial cells, a muscle cell, abrain cell, etc. Alternatively, the population of cells can be asubstantially homogeneous population, in which the population comprisesmainly of host cells (e.g., consisting essentially of) comprising therecombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein.

Included in the scope of the invention are conjugates, e.g.,bioconjugates, comprising any of the inventive polypeptides (includingany of the functional fragments or functional variants) orpeptidomimetics, nucleic acids, recombinant expression vectors, or hostcells. Conjugates, as well as methods of synthesizing conjugates ingeneral, are known in the art (See, for instance, Hudecz, F., MethodsMol. Biol. 298: 209-223 (2005) and Kirin et al., Inorg Chem. 44(15):5405-5415 (2005)).

The inventive polypeptides (including functional fragments andfunctional variants), peptidomimetics, fatty acid derivatives, nucleicacids, recombinant expression vectors, and host cells (includingpopulations thereof) can be isolated, purified, synthetic, and/orrecombinant. The term “isolated” as used herein means having beenremoved from its natural environment. The term “purified” as used hereinmeans having been increased in purity, wherein “purity” is a relativeterm, and not to be necessarily construed as absolute purity. Forexample, the purity can be at least about 50%, can be greater than 60%,70%, 80%, or 90%, or can be 100%.

The inventive polypeptides (including functional fragments andfunctional variants), peptidomimetics, fatty acid derivatives,conjugates, nucleic acids, recombinant expression vectors, and hostcells (including populations thereof), all of which are collectivelyreferred to as “inventive materials” hereinafter, can be formulated intoa composition, such as a pharmaceutical composition. In this regard, theinvention provides a pharmaceutical composition comprising any of thepolypeptides (including functional fragments and functional variants),peptidomimetics, fatty acid derivatives, conjugates, nucleic acids,recombinant expression vectors, and host cells (including populationsthereof), and a pharmaceutically acceptable carrier. The inventivepharmaceutical compositions containing any of the inventive materialscan comprise more than one inventive material, e.g., a polypeptide and anucleic acid, or two or more different polypeptides. Alternatively, thepharmaceutical composition can comprise an inventive material incombination with another pharmaceutically active agent or drug, such asa chemotherapeutic agent, e.g., asparaginase, busulfan, carboplatin,cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine,hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine,vincristine, etc.

In a preferred embodiment of the invention, the pharmaceuticalcomposition comprises the inventive material in combination with alipid. The lipid can be any lipid, including, for example, a fatty acid,a phospholipid, a sterol, a sphingolipid, a terpene, a glycerolipid, aglycerophospholipid, a prenol lipid, a saccharolipid, and a polyketide.Such lipids are known in the art. See, for example, Fahy et al., J.Lipid Res. 46: 839-861 (2005). Preferably, the lipid is a cholesterol.

With respect to pharmaceutical compositions, the pharmaceuticallyacceptable carrier can be any of those conventionally used and islimited only by chemico-physical considerations, such as solubility andlack of reactivity with the active compound(s), and by the route ofadministration. The pharmaceutically acceptable carriers describedherein, for example, vehicles, adjuvants, excipients, and diluents, arewell-known to those skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone which is chemically inert to the active agent(s) and one which hasno detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive material, as well as by the particular method used toadminister the inventive material. Accordingly, there are a variety ofsuitable formulations of the pharmaceutical composition of theinvention. The following formulations for oral, aerosol, parenteral,subcutaneous, intravenous, intramuscular, intraarterial, intrathecal,interperitoneal, rectal, and vaginal administration are exemplary andare in no way limiting. More than one route can be used to administerthe inventive materials, and in certain instances, a particular routecan provide a more immediate and more effective response than anotherroute. In a preferred embodiment of the invention, the pharmaceuticalcomposition is a topical formulation, an intravenous formulation, or asubcutaneous formulation.

In a preferred embodiment of the invention, the pharmaceuticalcomposition is a topical formulation. Topical formulations arewell-known to those of skill in the art. Such formulations areparticularly suitable in the context of the invention for application tothe skin. The topical formulation of the invention can be, for instance,a cream, a lotion, an ointment, a patch, an oil, a paste, a spray, e.g.,an aerosol spray, a gel, a mousse, a roll-on liquid, a solid stick, etc.Preferably, the topical formulation of the invention is a cream, alotion, an ointment, or a patch. When the topical formulation is alotion, preferably, the lotion also includes an ultraviolet (UV) lightblocking agent, such as tocopheryl, aminobenzoic acid, Avobenzone,Cinoxate, dioxybenzone, homosalate, menthyl anthranilate, octocrylene,octyl methoxycinnamate, octisalate, oxybenzone, padimate O,phenylbenzimidazole, sulfonic acid, sulisobenzone, titanium dioxide,trolamine salicylate, and zinc oxide.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the inventive materialdissolved in diluents, such as water, saline, or orange juice; (b)capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules;(c) powders; (d) suspensions in an appropriate liquid; and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol, and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant. Capsule forms can be of the ordinary hard- orsoft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers, such as lactose, sucrose, calciumphosphate, and corn starch. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise the inventive material in aflavor, usually sucrose and acacia or tragacanth, as well as pastillescomprising the inventive material in an inert base, such as gelatin andglycerin, or sucrose and acacia, emulsions, gels, and the likecontaining, in addition to, such excipients as are known in the art.

The inventive material, alone or in combination with other suitablecomponents, can be made into aerosol formulations to be administered viainhalation. These aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. They also may be formulated as pharmaceuticalsfor non-pressured preparations, such as in a nebulizer or an atomizer.Such spray formulations also may be used to spray mucosa.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The inventive material can be administered in a physiologicallyacceptable diluent in a pharmaceutical carrier, such as a sterile liquidor mixture of liquids, including water, saline, aqueous dextrose andrelated sugar solutions, an alcohol, such as ethanol or hexadecylalcohol, a glycol, such as propylene glycol or polyethylene glycol,dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain from about 0.5% toabout 25% by weight of the inventive material in solution. Preservativesand buffers may be used. In order to minimize or eliminate irritation atthe site of injection, such compositions may contain one or morenonionic surfactants having a hydrophile-lipophile balance (HLB) of fromabout 12 to about 17. The quantity of surfactant in such formulationswill typically range from about 5% to about 15% by weight. Suitablesurfactants include polyethylene glycol sorbitan fatty acid esters, suchas sorbitan monooleate and the high molecular weight adducts of ethyleneoxide with a hydrophobic base, formed by the condensation of propyleneoxide with propylene glycol. The parenteral formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampoulesand vials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid excipient, forexample, water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986)). Preferably, when administering cells, e.g., dendritic cells,the cells are administered via injection.

Additionally, the inventive materials, or compositions comprising suchinventive materials, can be made into suppositories by mixing with avariety of bases, such as emulsifying bases or water-soluble bases.Formulations suitable for vaginal administration can be presented aspessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the inventive materialsof the invention can be formulated as inclusion complexes, such ascyclodextrin inclusion complexes, or liposomes.

For purposes of the invention, the amount or dose of the inventivematerial administered should be sufficient to effect, e.g., atherapeutic or prophylactic response, in the subject or animal over areasonable time frame. For example, the dose of the inventive materialshould be sufficient to inhibit proliferation of a diseased cell, ortreat or prevent a disease (e.g., cancer, neoplasm, or psoriasis in aperiod of from about 2 hours or longer, e.g., 12 to 24 or more hours,from the time of administration. In certain embodiments, the time periodcould be even longer. The dose will be determined by the efficacy of theparticular inventive material and the condition of the animal (e.g.,human), as well as the body weight of the animal (e.g., human) to betreated.

Many assays for determining an administered dose are known in the art.For purposes of the invention, an assay, which comprises comparing theextent to which diseased cells are inhibited from proliferating, uponadministration of a given dose of an inventive material to a mammalamong a set of mammals of which is each given a different dose of theinventive material, could be used to determine a starting dose to beadministered to a mammal. The extent to which diseased cells areinhibited from proliferating upon administration of a certain dose canbe assayed by methods known in the art, including, for instance, themethods described herein as Example 2.

The dose of the inventive material also will be determined by theexistence, nature and extent of any adverse side effects that mightaccompany the administration of a particular inventive material.Typically, the attending physician will decide the dosage of theinventive material with which to treat each individual patient, takinginto consideration a variety of factors, such as age, body weight,general health, diet, sex, inventive material to be administered, routeof administration, and the severity of the condition being treated. Byway of example and not intending to limit the invention, the dose of theinventive material can be about 0.001 to about 1000 mg/kg body weight ofthe subject being treated/day, from about 0.01 to about 10 mg/kg bodyweight/day, about 0.01 mg to about 1 mg/kg body weight/day.

One of ordinary skill in the art will readily appreciate that theinventive materials of the invention can be modified in any number ofways, such that the therapeutic or prophylactic efficacy of theinventive materials is increased through the modification. For instance,the inventive materials can be conjugated either directly or indirectlythrough a linker to a targeting moiety. The practice of conjugatingcompounds, e.g., inventive materials, to targeting moieties is known inthe art. See, for instance, Wadwa et al., J. Drug Targeting 3: 111(1995) and U.S. Pat. No. 5,087,616. The term “targeting moiety” as usedherein, refers to any molecule or agent that specifically recognizes andbinds to a cell-surface receptor, such that the targeting moiety directsthe delivery of the inventive materials to a population of cells onwhich surface the receptor is expressed. Targeting moieties include, butare not limited to, antibodies, or fragments thereof, peptides,hormones, growth factors, cytokines, and any other natural ornon-natural ligands, which bind to cell surface receptors (e.g.,Epithelial Growth Factor Receptor (EGFR), T-cell receptor (TCR), B-cellreceptor (BCR), CD28, Platelet-derived Growth Factor Receptor (PDGF),nicotinic acetylcholine receptor (nAChR), etc.). The term “linker” asused herein, refers to any agent or molecule that bridges the inventivematerials to the targeting moiety. One of ordinary skill in the artrecognizes that sites on the inventive materials, which are notnecessary for the function of the inventive materials, are ideal sitesfor attaching a linker and/or a targeting moiety, provided that thelinker and/or targeting moiety, once attached to the inventivematerials, do(es) not interfere with the function of the inventivematerials, i.e., the ability to inhibit proliferation of a diseasedcell, or to treat or prevent disease (e.g., cancer, neoplasm,psoriasis).

Alternatively, the inventive materials can be modified into a depotform, such that the manner in which the inventive materials is releasedinto the body to which it is administered is controlled with respect totime and location within the body (see, for example, U.S. Pat. No.4,450,150). Depot forms of inventive materials can be, for example, animplantable composition comprising the inventive materials and a porousor non-porous material, such as a polymer, wherein the inventivematerials is encapsulated by or diffused throughout the material and/ordegradation of the non-porous material. The depot is then implanted intothe desired location within the body and the inventive materials arereleased from the implant at a predetermined rate.

It is contemplated that the inventive pharmaceutical compositions,polypeptides (including functional fragments and functional variants),peptidomimetics, fatty acid derivatives, nucleic acids, recombinantexpression vectors, host cells, or populations of cells can be used inmethods of inhibiting the proliferation of a diseased cell. In thisregard, the invention provides a method of inhibiting proliferation of adiseased cell. The method comprises contacting the diseased cell withany of the pharmaceutical compositions described herein in an amounteffective to inhibit proliferation of the diseased cell.

In a preferred embodiment of the host, the diseased cell is in a host.The host referred to herein can be any host. Preferably, the host is amammal. As used herein, the term “mammal” refers to any mammal,including, but not limited to, mammals of the order Rodentia, such asmice and hamsters, and mammals of the order Logomorpha, such as rabbits.It is preferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

The diseased cell can be a cell characteristic of or inflicted with anydisease. The disease can be any disease, condition, or malady,especially any of those caused by or involving the proliferation of acell. The disease can be, for example, a cancer or a non-canceroustumor, e.g., a cyst, a neoplasm, a fibroma, etc.

The cancer can be any cancer, including any of acute lymphocytic cancer,acute myeloid leukemia, a sarcoma, e.g., alveolar rhabdomyosarcoma, bonecancer, brain cancer, breast cancer, cancer of the anus, anal canal, oranorectum, cancer of the eye, cancer of the intrahepatic bile duct,cancer of the joints, glioma, cancer of the neck, gallbladder, orpleura, cancer of the nose, nasal cavity, or middle ear, cancer of theoral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronicmyeloid cancer, colon cancer, esophageal cancer, cervical cancer,gastrointestinal carcinoid tumor. Hodgkin lymphoma, hypopharynx cancer,kidney cancer, larynx cancer, liver cancer, lung cancer, malignantmesothelioma, melanoma, multiple myeloma, nasopharynx cancer,non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum,omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectalcancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestinecancer, soft tissue cancer, stomach cancer, testicular cancer, thyroidcancer, ureter cancer, and urinary bladder cancer. Preferably, thecancer is breast cancer, prostate cancer, ovarian cancer, stomach cancer(e.g., gastric adenocarcinoma), colon cancer, liver cancer, melanoma,basal cell carcinoma, rhabdomyosarcoma, medulloblastoma, pancreaticcancer, lung cancer, thyroid cancer, a myeloma, a lymphoma, a glioma, ora sarcoma.

As the proliferation of cells can cause a number of diseases, it isfurther contemplated that the inventive materials described herein canbe used in methods of treating or preventing these diseases. In thisregard, the invention provides a method of treating or preventing canceror a neoplasm (e.g., eye neoplasm) in a host. The method comprisesadministering to the host any of the pharmaceutical compositionsdescribed herein in an amount effective to treat the cancer or neoplasm.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer or a neoplasm in a mammal. Furthermore, the treatment orprevention provided by the inventive method can include treatment orprevention of one or more conditions or symptoms of the disease, e.g.,cancer, being treated or prevented. Also, for purposes herein,“prevention” can encompass delaying the onset of the disease, or asymptom or condition thereof.

In a preferred embodiment of the inventive methods, the pharmaceuticalcomposition is topically administered to the host. In another preferredembodiment, the pharmaceutical composition is administered directly tothe tumor, e.g., delivered intratumorally.

The invention furthermore provides a method of treating psoriasis in ahost comprising administering to the host any of the pharmaceuticalcompositions described herein in an amount effective to treat psoriasisin the host. Psoriasis is a common skin disease characterized bythickened patches of inflamed, red skin covered with thick, silveryscales. The psoriasis can be any form of psoriasis including, forexample, plaque psoriasis, or psoriasis vulgaris, pustular psoriasis,guttate psoriasis, and inverse psoriasis.

The invention also provides a method of inhibiting the Hedgehog signaltransduction pathway. The method comprises contacting the diseased cellwith any of the pharmaceutical compositions described herein in anamount effective to inhibit the Hedgehog signal transduction pathway.Since expression of certain genes are activated for transcription uponactivation of the Hedgehog signal transduction pathway, the inventionalso provides a method of inhibiting the expression of these genes in adiseased cell. The gene can be one or a combination of: Gli-1 (e.g.,GenBank Accession No. NM_(—)005269), Gli-2 (e.g., GenBank Accession No.NM_(—)005270), Gli-3 (e.g., GenBank Accession No._NM_(—)001034190), Ptch(e.g., GenBank Accession No. NM_(—)0000264), Shh (e.g., GenBankAccession No. NM_(—)000193), Smo (e.g., GenBank Accession No.NM_(—)005631), or NES (e.g., GenBank Accession No. NM_(—)016701), whichgenes are known in the art. The method of inhibiting the expression ofthese genes comprises contacting the diseased cell with any of thepharmaceutical compositions described herein in an amount effective toinhibit the expression of the gene.

For purposes herein, when a cell, e.g., a diseased cell is contactedwith a pharmaceutical composition comprising a nucleic acid orrecombinant expression vector, the method involves the expression of thenucleic acid such that the encoded polypeptide (or functional fragmentor functional variant) is expressed inside of the cell. When a cell,e.g., a diseased cell is contacted with a pharmaceutical compositioncomprising a host cell (or a population thereof), the method involvesthe expression of the nucleic acid inside of the host cell and thesecretion of the encoded polypeptide (or functional fragment orfunctional variant) outside of the host cell where the polypeptide isthen available to contact the diseased cell.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates a method of preparing polypeptides (includingfunctional fragments and functional variants) in accordance with anembodiment of the invention.

Polypeptides having the amino acid sequences as set forth in Table 1 aresynthesized by solid phase peptide synthesis on a 433A PeptideSynthesizer (Applied Biosystems, Foster City, Calif.) equipped with aconductivity monitoring unit utilizing Fmoc amino acid derivatives(AnaSpec, San Jose, Calif.). The synthesis is performed with conditionalblocking of unreacted amino groups with acetic anhydride for easierpurification of the resulting peptides. Peptides are cleaved from theresin with 87.5% trifluoroacetic acid containing 5% water, 5% thioanisoland 2.5% triisopropyl-silane, precipitated with cold diethyl ether,washed five times with ether and dried in vacuum overnight. Peptidesdissolved in dimethylformamide are purified by HPLC on a preparative(25×250 mm) Atlantis C18 reverse phase column (Agilent, Palo Alto,Calif.) in a gradient of 0.05% trifluoroacetic acid in water andacetonitrile containing 0.05% trifluoroacetic acid. The fractions areanalyzed by electrospray LC/MS on Agilent 1100 series instrument(Agilent Technologies, Palo Alto, Calif.) with the use of Zorbax300SB-C18 Poroshell column and a gradient of 5% acetic acid in water andacetonitrile. Only fractions containing more than 95% pure product arecombined and freeze-dried. Peptides are dried from 5% acetic acid toensure conversion into acetate salts. The purity and structure arefurther confirmed by LC/MS with separation on Zorbax 300SB-C18analytical column.

TABLE 1 SEQ Peptide ID Name Amino acid sequence NO: SMO-i3-1PalRGVMTLFSIKSNHPGLLSEKAASKINETMLR 4 SMO-i3-2 PalRGVMTLFSIKSNHPGLLSEKA 9SMO-i3-4 PalLFSIKSNHPGLLSEKAASKINETMLR 10 SMO-i3-5RGVMTLFSIKSNHPGLLSEKAASKINETMLRK-ε □Pal 60 SMO-i3-6 LLSEKAASKINETMLRK-□ε-Pal 61 SMO-i3-7 LFSIKSNHPGLLSEKAASKINETMLRK-□ ε-Pal 62 SMO-i3-8PalRGVMTLFSIKSNHPGLLS 14 SMO-i3-9 PalHseARGVMTLFSIKSNHPGLLS 77 SMO-i3-10PalRGVMTLFSIKSNH 15 SMO-i3-12 SEKAASKINETMLRK-□ ε-Pal 63 SMOi2-1PalLTYAWHTSFKALGTTYQPLSGKYSY 3 SMOi2-2 PalLTYAWHTSFKALGTTYQPLSGKTSY 17SMOi2-3 PalLTYAWHTSFKALGTTYQPLSG 18 SMOi2-4AcLTYAWHTSFKALGTTYQPLSGKTSYK-ε-Pal 64 SMOi2-5AcYAWHTSFKALGTTYQPLSGKTSYK-□ ε-Pal 65 SMOi2-6 PalLTYAWHTSFKALGTTYQP 21SMOi2-7 GTTYQPLSGKTSYK-□ ε-Pal 66 SMOi2-8 PalLTYAWHTSFKAL 23 SMOi2-9AcLTYAWHTSFKAL 24 SMOi2-10 PalTYAWHTSFKAL 25 SMOi2-11 PalLTYAWHTSFKA 26SMOi2-12 PalLTYAWHTSFK 27 SMOi2-13 AcTYAWHTSFKA 28 SMOi2-14VWFVVLTYAWHTSFKAL 55 SMOi2-15 WFVVLTYAWHTSFKAL 56 SMOi2-16AcLAKFSTHWAYTLK(ε-Pal)-All-D 67 SMOi2-17 AcAKFSTHWAYTLK(ε-Pal)-All-D 68SMOi2-18 PalLTYABpaHTSFKAL 54 SMOi2-20 AcKFSTHWAYTLK(ε-Pal)All-D 69SMOi2-21 Pal-LTYABpaHTSFKAL-Hcy-Biotin 81 SMOi2-22 AKFSTHWAYTL (All-D)37 SMOi2-23 PalLTYAWHTSFKALGTTYQPLSGKTSYK(ε-Pal) 70 SMOi2-24PalLTYAWHTSFKAL (All-D) 30 SMOi2-25 AcLTYAWHTSFKAL (All-D) 31 SMOi2-26MyrLTYAWHTSFKAL 32 SMOi2-29 Ac-LTYAWHTSFKAL-Penetratin 82 SMOi2-30Penetratin-LTYAWHTSFKAL 83 SMOi2-56

84 SMOi2-57 D-(LAKFSTHWAYTL)-K-(ε-Pal)-LTYAWHTSFKAL 92 SMOi2-58D-(AKFSTHWAYTL)-K-(ε-Pal)-LTYAWHTSFKAL 93 SMOi2-59D-(AKFSTHWAYTL)-K-(ε-Pal)-LTYAWHTSFKA 94 Pal = palmitate; “all D” = eachamino acid of the polypeptide is the D isomer; Ac = acetate; Myr =myristate; PalHse = homoserine palmitate; Bpa = 4-benzoylphenylalanine;and HCy-Biotin, homocysteine-Biotin, in which biotin is attached to theSH of homocysteine.

The peptides described herein can be made into a dimeric form having thefollowing general structure:

wherein Sequence X is selected from the group consisting ofAc-LAKFSTHWAYTL (all-D) (SEQ ID NO: 85); Ac-AKFSTHWAYTL (All-D) (SEQ IDNO: 86); and Ac-KFSTHWAYTL (All-D) (SEQ ID NO: 87); wherein each ofLinker 1 and Linker 2 is optionally present and each independently isGly, beta-Ala, aminopropionic acid, gamma-aminobutyric acid,aminocaproic acid, or aminohexanoic acid; wherein n and m is between 0and 6; wherein Y is K, C, homoCys, Orn, diaminopropanoic acid (DPA),diaminobutyric acid (DBA); and wherein the fatty acid is a stearic,palmitic, myristic, lauric, capric or caprilic acid. In a preferredembodiment, Sequence X is SEQ ID NO: 87, n is 4, m is 0, each of Linker1 and Linker 2 is beta-Ala, and the fatty acid is palmitate.

For the synthesis of such dimeric inhibitors, wherein Y is Lys, resinpreloaded with Fmoc-Lys with fatty acid attached to the c-amino group isreacted with a corresponding diamino acid (e.g., Orn, Lys,diaminobutyric acid (DBA), or diaminopropionic acid (DPA)) that has anFmoc protection group on one amino group and a DDE protection group onthe other amino group. The DDE group is selectively removed with amixture of hydroxylamine and imidazole in DMF. The resulting resin iscoupled to a linker amino acid (e.g., Fmoc-Gly, beta-Ala, aminopropionicacid, gamma-aminobutyric acid, aminocaproic acid, or aminohexanoic acid)on an ABI433 peptide synthesizer. The remainder of each Sequence X issimultaneously built on a peptide synthesizer using a standard syntheticprotocol. The dimeric product is cleaved, deprotected and purified as ina standard synthetic protocol. SMOi2-56, which is the dimeric form ofSMOi2-17, is made in this manner.

SMOi2-29 and -30 are peptides based on SMOi2-9 fused to penetratin,which is a peptide from Antennapedia used to introduce a variety ofbiologically active molecules, such as DNA, peptides, or proteins intocells (Granier et al., J. Biol. Chem. 279: 50904-50914 (2004)).Penetratin has the amino acid sequence RQIKIWFPNRR-Nle-KWKK (SEQ ID NO:78). Penetratin-containing peptides are made as a single peptide chainusing standard peptide synthesis methods.

Polypeptides are lipidated as follows: for L-peptides containings-palmitoyl-Lys on the C-terminus, commercially availableFmoc-s-palmitoyl-L-Lys (AnaSpac, San Jose, Calif.) is utilized.Fmoc-s-palmitoyl-D-Lys is not commercially available. It is synthesizedon the resin utilizing orthogonally protected Fmoc-D-Lys(ivDDE)(N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-D-lysine)(Novabiochem, San Diego, Calif.). After attachment of the amino acid toRink-amide resin, ivDDE protection group is removed by treatment withhydrazine/imidazole mixture in NMP. The resin is washed with NMP andreacted with 10-fold excess of palmitic acid/HBTU/HOBt in NMP for twohours. After washing of the resin with NMP, the synthesis is continuedutilizing standard protocols on the peptide synthesizer. For peptidescomprising myristic acid or acetate at the N-terminus, the correspondingfatty acid (10-fold excess) was dissolved in NMP or NMP/DCM mixture,activated with HBTU/HOBt mixture and reacted with the peptide on theresin. Subsequent cleavage and deprotection was carried out as was donefor lipidations with palmitic acid.

The molecular mass of each peptide is determined by ion-spray massspectrometry utilizing an Agilent1100 LC/MS system (Agilent, SantaClara, Calif.) and is shown in Tables 2 and 3.

TABLE 2 Mass Mass Compound (calculated) (found) t_(R) ^(a)(min) PuritySMO i2-1 3121.7 3121.0 16.67 95% SMO i2-2 3059.6 3059.0 16.51 95% SMOi2-3 2580.0 2580.0 17.13 96% SMO i2-4 3015.5 3015.0 16.12 95% SMO i2-53229.8 3230.0 16.34 95% SMO i2-6 2225.6 2225.5 17.13 96% SMO i2-7 1646.91647.0 18.48 98% SMO i2-8 1675.1 1675.0 17.59 96% SMO i2-9 1478.6 1478.014.64 100%  SMO i2-10 1561.9 1562.0 18.65 98% SMO i2-11 1561.9 1562.018.99 98% SMO i2-12 1490.8 1491.0 19.04 97% SMO i2-13 1294.4 1294.013.97 99% SMO i2-14 2109.4 2109.0 18.46 96% SMO i2-15 2010.3 2010.017.86 96% SMO i2-16 1845.2 1845.0 18.96 97% SMO i2-17 1732.1 1732.018.63 97% SMOi2-18 1740.1 1740.0 17.75 96% SMOi2-20 1661.0 1661.0 17.1198% SMOi2-21 2292.3 2292.0 17.08 96% SMOi2-23 3426.2 3426.0 18.00 95%SMOi2-24 1675.1 1674.9 17.01 96% SMOi2-25 1477.9 1477.9 13.85 98%SMOi2-29 3689.4 3689.0 13.95 95% ^(a)The retention times are for Zorbax300SB-C3 column (Agilent, Santa Clara, CA) determined in 0-100% 25 mingradient of 0.5% acetic acid in water and 0.5% acetic acid inacetonitrile, flow rate of 0.3 mL per minute.

TABLE 3 Mass Mass Compound (calculated) (found) t_(R) ^(a)(min) PuritySMOi3-1 3630.9 3630.9 16.08 95% SMOi3-2 2504.1 2504.4 16.42 96% SMOi3-43104.8 3104.4 16.69 95% SMOi3-5 3801.0 3801.0 15.87 95% SMOi3-6 2193.42193.4 15.26 96% SMOi3-7 3274.9 3274.5 16.08 95% SMOi3-8 2176.7 2176.217.05 96% SMOi3-10 1709.1 1708.8 17.23 97% SMOi3-12 1967.4 1967.2 16.0997% ^(a)The retention times are for Zorbax 300SB-C3 column (Agilent,Santa Clara, CA) determined in 0-100% 25 min gradient of 0.5% aceticacid in water and 0.5% acetic acid in acetonitrile, flow rate of 0.3 mLper minute.

HPLC of the peptides is performed on a Microsorb-MW 300A C8 column(Varian, Palo Alto, Calif.) in 0-100% 20 min gradient of 0.1%trifluoroacetic acid in water/acetonitrile containing 0.1%trifluoroacetic acid, flow rate 1 ml/min. Peptides are detected by UVmonitoring at 225, 256, and 280 nm. Data not shown.

Example 2

This example demonstrates a method of testing the inventive polypeptidesfor toxicity.

DU145 prostate cancer cells, PC3 prostate cancer cells, MCF7 breastcancer cells, or MeI-SK-2 melanoma cells (American Type CultureCollection, Manassas, Va.) are inoculated in 96 well plates at 200-400cells/well density in DMEM medium containing 10% fetal bovine serum andallowed to attach for 24 hours. Cell suspension of 100 μl is used foreach well. Polypeptides in 100 μl medium at 2× concentration are addedthe next day and kept in the CO₂ incubator for 48 hours. While thepolypeptides are added at a final concentration between 1 nM and 10 μM,assays are performed on extra reference plates to determine the cellpopulation density at time 0 (T₀). The cells are stained with PromegaNon-Radioactive Cell Proliferation Assay Kit (MTT) according tomanufacture's protocol. The absorbance of the wells is determined at 544nm by a FLUOstar/POLARstar® Galaxy MicroplateReader (BMG LabtechnologiesGmbH, Germany). The assays are performed on control (C) and test (T)cells. Cellular responses are calculated from the data using thefollowing formula: 100×[(T−T₀)/(C−T₀)] for T>T₀ and 100×[(T−T₀)/T₀] forT<T₀.

Example 3

This example demonstrates that polypeptides in accordance withembodiments of the invention are able to inhibit proliferation ofdiseased cells.

Polypeptides corresponding to the full lengths of all threeintracellular loops of SMO (SMOi1-1, SMOi2-1, SMOi3-1) having anN-terminal palmitoyl residue are constructed as described in Example 1.The polypeptides are then tested for toxicity (growth inhibition) asdescribed in Example 2 using MCF-7 breast cancer cells and gastricadenocarcinoma cells. Activity of SMOi2-1 and SMOi3-1 is compared tothat of cyclopamine (5 μM), a teratogen isolated from the corn lilyVeratrum califonicum.

As shown in FIG. 1, all three peptides inhibit the growth of MCF-7cells. The SMOi3-1 polypeptide has the most significant effect on cellgrowth, followed by SMOi2-1, while SMOi1-1 demonstrates the least amountof inhibitory activity. As shown in FIG. 2, SMOi3-1 and SMOi2-1polypeptides are able to inhibit the growth of gastric adenocarcinomacells as well or better than cyclopamine.

Example 4

This example demonstrates that functional fragments and functionalvariants having an amino acid sequence based on the second and thirdintracellular loops of the SMO protein in accordance with an embodimentof the invention are able to inhibit proliferation of diseased cells.

Polypeptides based on the second or third intracellular loop of SMO(SMOi2 or i3 polypeptides) (as shown in Table 1) are synthesized asdescribed in Example 1 and are tested as described in Example 2 usingMCF-7 breast cancer cells or SK-Mel2 melanoma cells. The IC₅₀ of eachpeptide as determined by the MTT assay in SK-Mel2 melanoma cells after48 hour exposure to the peptide is shown in Tables 4 and 5.

TABLE 4 Compound Structure IC₅₀, μM SMO i2-1Pal-LTYAWHTSFKALGTTYQPLSGKYSY 0.45 ± 0.05 SMO i2-2Pal-LTYAWHTSFKALGTTYQPLSGKTSY 0.45 ± 0.05 SMO i2-3Pal-LTYAWHTSFKALGTTYQPLSG 1.4 ± 0.4 SMO i2-4Ac-LTYAWHTSFKALGTTYQPLSGKTSYK-ε-Pal 1.0 ± 0.1 SMO i2-5Ac-YAWHTSFKALGTTYQPLSGKTSYK-ε Pal 1.0 ± 0.1 SMO i2-6Pal-LTYAWHTSFKALGTTYQP  0.3 ± 0.05 SMO i2-7 Ac-GTTYQPLSGKTSYK-ε Pal 2.7± 0.4 SMO i2-8 Pal-LTYAWHTSFKAL 0.08 ± 0.02 SMO i2-9 Ac-LTYAWHTSFKAL >10SMO i2-10 Pal-TYAWHTSFKAL 0.7 ± 0.1 SMO i2-11 Pal-LTYAWHTSFKA  0.09 ±0.007 SMO i2-12 Pal-LTYAWHTSFK  0.06 ± 0.007 SMO i2-13 Ac-TYAWHTSFKA 2.8± 0.3 SMO i2-14 VWFVVLTYAWHTSFKAL  >5 SMO i2-15 WFVVLTYAWHTSFKAL  >5 SMOi2-16 Ac-LAKFSTHWATYLK-ε-Pal (all D-)  0.006 ± 0.0005 SMO i2-17Ac-AKFSTHWATYLK-εPal (all D-) 0.0004 ± 0.0001 SMO i2-18Pal-LTYABpaHTSFKAL  0.1 ± 0.05 SMO i2-20 Ac-KFSTHWATYLK-εPaL (all D-)0.0003 ± 0.0001 SMO i2-21 Pal-LTYABpaHTSFKAL-Hcy-Biotin >15 SMO i2-23Pal-LTYAWHTSFKALGTTYQPLSGKTSYK-ε-Pal 0.05 ± 0.02 SMOi2-24PalLTYAWHTSFKAL (All D) 0.039 ± 0.004 SMOi2-25 AcLTYAWHTSFKAL (AllD) >10 SMO i2-26 Myr-LTYAWHTSFKAL  0.2 ± 0.05 SMO i2-29Ac-LTYAWHTSFKAL-Penetratin >15 SMO i2-30 Penetratin-LTYAWHTSFKAL >15SMOi2-56

4.0 nm Pal, palmitate; Ac, acetate; (All D), each amino acid of thepolypeptide is the D isomer; Myr, myristic acid; ε-Pal, palmitate addedon the ε carbon of Lys; (Bpa), 4-benzoylphenylalanine.

TABLE 5 Compound Structure IC_(50,)μM SMO i3-1PalRGVMTLFSIKSNHPGLLSEKAASKINETML 0.64 ± 0.1 SMO i3-2PalRGVMTLFSIKSNHPGLLSEKA 0.50 ± 0.1 SMO i3-4PalLFSIKSNHPGLLSEKAASKINETMLR  1.5 ± 0.2 SMO i3-5AcRGVMTLFSIKSNHPGLLSEKAASKINETMLRK- ε -Pal  0.9 ± 0.2 SMO i3-6Ac-LLSEKAASKINETMLRK-□ε-Pal  0.8 ± 0.1 SMO i3-7Ac-LFSIKSNHPGLLSEKAASKINETMLRK-□ ε -Pal 0.95 ± 0.2 SMO i3-8PalRGVMTLFSIKSNHPGLLS  0.5 ± 0.1 SMO i3-10 PalRGVMTLFSIKSNH 0.95 ± 0.2SMO i3-12 Ac-LLSEKAASKINETMLRK□ ε -Pal 1.33 ± 0.2

As shown in Table 5 and FIG. 3, polypeptides based on the thirdintracellular loop of SMO exhibit the ability to inhibit the growth ofMCF-7 cells. Also, peptides corresponding to fragments of the thirdintracellular loop have activities that are comparable or lower than thefull-length loop (SMOi3-1).

As shown in Table 4, several of the polypeptides based on the secondintracellular loop of SMO (SMOi2 polypeptides) are able to inhibit thegrowth of SK-Mel2 melanoma cells after 48 hours of exposure to thepolypeptides. Among the most potent inhibitors are SMOi2-16, SMOi2-17,SMOi2-8, SMOi2-23, SMOi2-24, SMOi2-20, SMOi2-26, SMOi2-11, and SMOi2-12.Also, SMOi2-6, SMOi2-7, SMOi2-2 through SMOi2-5, SMOi2-10, and SMOi2-13are potent inhibitors. C-terminal truncation of the second intracellularloop yields polypeptides that were significantly more toxic to cancercells than the full-length loop (SMOi2-1). Both halves of the loop whenpalmitoylated at the amino acids which are positioned at the end of theloop (which end is adjacent to the membrane in the wild-type SMOprotein) are active. However, C-terminal extension of the N-terminalhalf lowers the activity of the most potent 12-residue long polypeptide(compare peptides SMOi2-8 with SMOi2-6 and SMOi2-3).

Palmitic acid lipidation of the polypeptides appears to be essential forthe activity, since substitution of palmitate with an acetyl residuesignificantly reduces the inhibitory activity (compare SMOi2-9 andSMOi2-13). This is likely due to poor cell penetration of thepolypeptide. It appears necessary for the palmitoylation to occur at theend of the loop (which end is adjacent to the membrane in the wild-typeSMO protein), since positioning the palmitoyl group inside the loopgenerated a significantly less active peptide (SMOi3-4 of Table 5). Thegrowth inhibition curves of these polypeptides either plateau at higherconcentrations or curve upward, indicating that inhibitory activityactually decreases at higher concentrations. SMOi3-1, SMOi3-8, SMOi3-2,SMOi3-6, and SMOi3-12 are among the most potent polypeptides tested.

Unlike SMOi3 polypeptides, all second loop derivatives have “normal”concentration-dependence profiles of growth inhibition activity.

As an alternative delivery of the peptides inside the cells, SMOi2-9 isfused to penetratin. Neither C-terminal nor N-terminal fusion helps torestore the activity, suggesting that palmitoylation provides more thanjust cell permeability. Also, the replacement of palmitoyl residue withsequences of the transmembrane domain of the SMO protein does notovercome the loss of activity (SMOi2-14 and SMOi2-15). The lack ofactivity may be due to the fact that these polypeptides have poorsolubility. Substitution of palmitoyl residue with slightly shortermyristoyl resulted in 2.5-fold less potent compound (SMOi2-26). Forstudying peptide localization inside the cells and characterization ofthe interacting protein molecules, synthesis of cross-linkablederivative labeled with biotin is attempted. Substitution of Trp residueof SMOi2-8 with p-benzoyl-phenylalanine that can be UV cross-linked to aprotein ligand produces a fairly active compound (SMOi2-18, Table 4).However, addition of maleimide-biotin coupled through SH-group ofC-terminal homocysteine (SMOi2-21) totally abolishes the activity, thusrendering it unsuitable for receptor identification.

The retroinverso analogues of SMOi2 polypeptides exhibit inhibitoryactivity. Both SMOi2-16, which is the retroinverso analogue of SMOi2-8,and its truncated version SMOi2-17, which is the retroinverso analogueof SMOi2-11 (and SMOi2-12), are more potent in inhibiting and killingmelanoma cells than their all-L parent polypeptide (FIGS. 4 and 7).

Example 5

This example demonstrates a method of inhibiting the gene expression ofHedgehog signaling pathway proteins in cells in accordance with anembodiment of the invention.

An analysis of expression of the genes that are known markers of theHedgehog signal transduction pathway, Gli-1, Gli-2, Gli3, Ptch, Shh, Smoand NES is performed. DU145 prostate cancer cells are exposed to SMOi3-1(5 or 10 μM), SMOi2-1 (5 or 10 μM) or cyclopamine (5 μM) for 48 hours.Gene expression is assayed by quantitative PCR. For gene expressionassay, DU145 prostate cancer cells were exposed to 5 μM SMOi3-1 for 24h, and 5 μM and 10 μM SMOi3-1 for 48 h, respectively. DU145 cells weretreated by 5 μM and 10 μM of SMOi2-1 for 24 h only. The control wasDU145 cells without compounds-treatment. The 5 μM of cyclopamine wasalways used as positive control in all experiments.

Total cellular RNA was isolated, and further purified by RNeasy® columns(QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.RNA quality and quantity were determined using Agilent RNA 6000 NanoChip (Agilent Technologies, Inc., CA). cDNA synthesis was carried outusing Random Hexamer primer, TaqMan® Reverse Transcription Reagents kit(Applied Biosystems, Foster, Calif.).

Taqman® Gene Expression Assay primer and probe (FAM-labeled) sets(Applied Biosystems, Foster, Calif.) are used for real-time quantitativePCR analysis of PTCH (Assay ID=Hs00181117_ml), GUI (Hs00171790_ml), GLI2(Hs00257977_ml), GLI3 (Hs00609233_ml), SMO (Hs00170665_ml), SHH(Hs00179843_ml) and NES (Hs00707120_s1). TaqMan® Gene Expression Assaymix of primer and probe (VIC-labeled) of 18S rRNA was used as theendogenous control. Each sample is run in triplicate. Triplicate Ctvalues were analyzed using the comparative Ct (ΔΔCt) method as describedby the manufacturer (Applied Biosystems, Foster, Calif.). The relativeamount of target (2^(−ΔΔCt)) is obtained by normalization to anendogenous reference (18s rRNA).

As shown in FIG. 5, the changes in gene expression are more pronouncedthan that of SMO antagonist, cyclopamine. This is consistent with thefact that the polypeptides have a much higher potency than cyclopamine.

Example 6

This example demonstrates that inventive peptidomimetics in accordancewith embodiments of the invention inhibit the proliferation of diseasedcells.

Peptides SMOi2-18 and SMOi2-21 are made as essentially described inExample 1 and tested as described in Example 2 using SK-Mel2 cells.Cells are exposed to polypeptides or peptidomimetics for 60 hours.

As shown in FIG. 6, peptidomimetics, SMOi2-18 and SMOi2-21, each ofwhich contains the synthetic amino acid, BPA, exhibit the ability toinhibit the proliferation of SK-Mel2 cells. The inhibitory activity iseven more potent than that of the polypeptide counterpart, SMOi2-8,which contains only naturally-occurring amino acids.

Example 7

This example demonstrates the critical micelle concentration of SMOi2-8.

To monitor the formation of hydrophobic nanoparticles, a fluorescingimidazoacridone compound WMC-77(5-{3-[4-(aminopropyl)-piperazin-1-yl]-propylamino}-2,10b-diaza-aceanthrylen-6-one)is used (Tarasov et al., Photochem. Photobiol. 78: 313-322 (2003)).Compounds like WMC-77 tend to enhance their intrinsic fluorescencedramatically when entering an amphiphilic environment of biologicalmacromolecules like DNA (Tarasova et al., 2003, supra) or hydrophobiccore of typical micelles (Tarasov et al., Photochem. Photobiol. 70:568-578 (1999)). Since imidazoacridones are adsorbed on quartz from theaqueous solutions, plastic polymethacrylate 10×10 mm cells fromSigma-Aldrich (St. Louis, Mo.) are used for most measurements.Atmospheric oxygen quenching is found to be unimportant, since similarvalues of fluorescence intensity from BIAs are obtained before and afternitrogen purging. The solutions are prepared in deionized water.Uncorrected fluorescence emission spectra are obtained at 25° C. on aSingle Photon Counting Spectrofluorometer FLUOROMAX®-2 (Horiba JobinYvon, Edison, N.J.). The excitation and emission monochromator slits areadjusted to 1.5 and 3.5 nm bandwidth, respectively. The emission spectra(increment 1 nm, integration time 0.2 sec.) are collected at the range450-700 nm, using 430 nm excitation monochromator setting. Thefluorometric measurements are performed for premixed aliquots of peptideand imidazoacridone solutions. The concentration of fluorescing agentWMC-77 in all probes is 0.4 μM.

The fluorescence data are presented in FIG. 8. The increase ofpeptide/fluorophore ratio causes the permanent increase of WMC-77fluorescence emission intensity, leveling of at ˜2 μM. The changes inthe fluorescence signal are similar to those observed during transfer ofimidazoacridones from aqueous to non-polar media such as organicsolvents (Tarasov et al., 1999, supra), to the cores of classicsurfactant micelles (Tarasov et al., 1999, supra) or upon binding to DNA(Tarasov et al., 2003, supra). The critical micelle concentration,estimated as described in (Tanford, The Hydrophobic Effect: Formation ofMicelles and Biological Membranes, John Wiley & Sons, New York (1980)),is determined as 0.5-1 μM of SMOi2-8.

Critical micelle concentration is tested as described above for allother peptides of this study and is found to be around 1 μM for eachpeptide. Micellization may be responsible for lowering the effectiveconcentration of free peptides in solution and subsequent apparentreduction in potency. The majority of peptides also precipitated out inmedium at concentration higher than 10 μM.

Example 8

This example demonstrates that the peptides of the invention exhibitdifferent sensitivities toward different cell lines.

Cancer cells of the breast (T47D), melanoma (SK-MEL-2), hepatoma (HepG2,PLC, JM-1), pancreas (Panc10.05, HS766T), colon (Colo205, HCT15), andlung (A549) are cultured in medium containing either SMOi2-12 orSMOi2-20 at a concentration of 0.001, 0.01, 0.1, 1.0, or 5 μM. Cells areassayed as described in Example 2. As shown in FIG. 9, SMOi2-12 andSMOi2-20 exhibit different sensitivities depending on the cell treated.Cell lines exhibiting a GI₅₀>5 include PLC, JM-1, HS766T, Colo205,HCT15, and A549.

Example 9

This example demonstrates the peptides of the invention have secondarystructure.

The peptides are measured by circular dichroism (CD) spectroscopy.Peptide solutions (1 μM) are prepared by dissolving compounds in PBScontaining 50 mM dodecylphophocholine (Avanti Polar Lipids, Alabaster,Ala.). CD spectra are recorded by an AVIV mod. 202 CD-spectrometer (AvivInstruments, Lakewood, N.J.) using 0.1 cm path length quartz cuvette at22-24° C. Scan ranges are between 180 and 260 nm and the spectrum of thebuffer is subtracted from the spectrum of the compound.

The CD spectra of SMOi2-8 and SMOi2-16 (FIG. 10) demonstrate that thepeptides predominantly adopt a beta-strand conformation. Theretro-inverso peptides appear to be more structured and rigid than theparent all-L counterparts.

Example 10

The example demonstrates alanine scanning studies of the polypeptides ofthe invention.

The significance of different residues in the SMOi2-8 sequence(PalLTYAWHTSFKAL) is probed by creating a collection of mutants of theSMOi2-8 peptides in which each mutant of the collection has an aminoacid residue substituted with Ala and every residue of SMOi2-8 istargeted for mutation by at least one of the mutants in the collection.

The Lys residue at the 10th position of SMOi2-8 is critical for theactivity of the SMOi2-8 peptide. Significant loss in activity also isobserved upon substitution of the Ser at position 8 of SMOi2-8. Activityincreased when the Leu at position 1 was replaced with Ala. The Phe atposition 9, Tyr at position 3, and Trp at position 5 can be substitutedwith Ala without significant change in the activity. The remainingsubstitutions (2, 4, 6, 7, 11, and 12) result in a slight (40-60%)increase in GI₅₀

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1-32. (canceled)
 33. A method of inhibiting proliferation of a diseasedcell, the method comprising contacting the diseased cell with apharmaceutical composition comprising (i) an isolated or purifiedpolypeptide, peptidomimetic, or fatty acid derivative of the polypeptideor peptidomimetic comprising an amino acid sequence comprising SEQ IDNO: 3, a functional fragment thereof, or a functional variant of eitherSEQ ID NO: 3 or the functional fragment, wherein the functional variantcomprises an amino acid sequence (a) which has at least 90% sequenceidentity with SEQ ID NO: 3 or (b) which is a retroinverso analogue ofSEQ ID NO: 3 or a functional fragment thereof, wherein the polypeptideor peptidomimetic is less than or equal to 50 amino acids in length,wherein the functional fragment comprises at least 7 contiguous aminoacids of SEQ ID NO: 3, and wherein the functional fragment or functionalvariant inhibits proliferation of a diseased cell, (ii) optionally alipid, and a (iii) pharmaceutically acceptable carrier in an amounteffective to inhibit proliferation of the diseased cell.
 34. The methodof claim 33, wherein the diseased cell is in a host.
 35. The method ofclaim 34, wherein the host is a mammal.
 36. The method of claim 35,wherein the mammal is a human.
 37. The method of claim 33, wherein themethod treats or prevents a cancer of the host.
 38. The method of claim37, wherein the cancer is breast cancer, prostate cancer, ovariancancer, stomach cancer, colon cancer, liver cancer, melanoma, basal cellcarcinoma, rhabdomyosarcoma, a medulloblastoma, pancreatic cancer, lungcancer, thyroid cancer, a myeloma, a lymphoma, a glioma, or a sarcoma.39. The method of claim 38, wherein the stomach cancer is a gastricadenocarcinoma.
 40. The method of claim 33, wherein the method treats orprevents a neoplasm of the host.
 41. The method of claim 33, wherein thepharmaceutical composition is topically administered to the host. 42.The method of claim 33, wherein the pharmaceutical composition isintratumorally administered to the host.
 43. A method of treating orpreventing cancer, psoriasis, or a neoplasm in a host, the methodcomprising administering to the host a pharmaceutical compositioncomprising (i) an isolated or purified polypeptide, peptidomimetic, orfatty acid derivative of the polypeptide or peptidomimetic comprising anamino acid sequence comprising SEQ ID NO: 3, a functional fragmentthereof, or a functional variant of either SEQ ID NO: 3 or thefunctional fragment, wherein the functional variant comprises an aminoacid sequence (a) which has at least 90% sequence identity with SEQ IDNO: 3 or (b) which is a retroinverso analogue of SEQ ID NO: 3 or afunctional fragment thereof, wherein the polypeptide or peptidomimeticis less than or equal to 50 amino acids in length, wherein thefunctional fragment comprises at least 7 contiguous amino acids of SEQID NO: 3, and wherein the functional fragment or functional variantinhibits proliferation of a diseased cell, (ii) optionally a lipid, anda (iii) pharmaceutically acceptable carrier in an amount effective totreat or prevent the cancer, psoriasis, or a neoplasm. 44-45. (canceled)46. A method of inhibiting the expression of a gene selected from thegroup consisting of Gli-1, Gli-2, Gli-3, Ptch, Shh, Smo, NES, and acombination thereof, in a diseased cell, the method comprisingcontacting the diseased cell with a pharmaceutical compositioncomprising (i) an isolated or purified polypeptide, peptidomimetic, orfatty acid derivative of the polypeptide or peptidomimetic comprising anamino acid sequence comprising SEQ ID NO: 3, a functional fragmentthereof, or a functional variant of either SEQ ID NO: 3 or thefunctional fragment, wherein the functional variant comprises an aminoacid sequence (a) which has at least 90% sequence identity with SEQ IDNO: 3 or (b) which is a retroinverso analogue of SEQ ID NO: 3 or afunctional fragment thereof, wherein the polypeptide or peptidomimeticis less than or equal to 50 amino acids in length, wherein thefunctional fragment comprises at least 7 contiguous amino acids of SEQID NO: 3, and wherein the functional fragment or functional variantinhibits proliferation of a diseased cell, (ii) optionally a lipid, anda (iii) pharmaceutically acceptable carrier in an amount effective toinhibit the expression of the gene.
 47. A method of inhibiting theHedgehog signal transduction pathway in a diseased cell, the methodcomprising contacting the diseased cell with a pharmaceuticalcomposition comprising (i) an isolated or purified polypeptide,peptidomimetic, or fatty acid derivative of the polypeptide orpeptidomimetic comprising an amino acid sequence comprising SEQ ID NO:3, a functional fragment thereof, or a functional variant of either SEQID NO: 3 or the functional fragment, wherein the functional variantcomprises an amino acid sequence (a) which has at least 90% sequenceidentity with SEQ ID NO: 3 or (b) which is a retroinverso analogue ofSEQ ID NO: 3 or a functional fragment thereof, wherein the polypeptideor peptidomimetic is less than or equal to 50 amino acids in length,wherein the functional fragment comprises at least 7 contiguous aminoacids of SEQ ID NO: 3, and wherein the functional fragment or functionalvariant inhibits proliferation of a diseased cell, (ii) optionally alipid, and a (iii) pharmaceutically acceptable carrier in an amounteffective to inhibit the Hedgehog signal transduction pathway. 48-50.(canceled)
 51. The method of claim 33, wherein the polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:17 to
 33. 52. The method of claim 33, wherein the functional variantcomprises the amino acid sequence of SEQ ID NO: 23 with one amino acidsubstitution at any of positions 1-7,9,11, and 12 of SEQ ID NO: 23 inwhich the amino acid at the position is substituted with Ala.
 53. Themethod of claim 33, wherein the functional variant comprises aretroinverso analogue of SEQ ID NO: 23, 26, or
 33. 54. The method ofclaim 33, wherein the functional variant comprises the amino acidsequence of any of SEQ ID NOs: 34 to 37, 68, 84, and 92-94.
 55. Themethod of claim 33, wherein the functional variant comprises the aminoacid sequence of any of SEQ ID NOs: 38-59.
 56. The method of claim 33,wherein the fatty acid derivative comprises a fatty acid molecule at theamino (N-) terminus, the carboxyl (C-) terminus, or both the N- andC-termini, the fatty acid molecule optionally containing at least oneamino group.
 57. The method of claim 33, wherein the functional variantcomprises the amino acid sequence of SEQ ID NO: 68 (all D-amino acids),the functional variant is optionally amidated, and the amino acid atposition 1 of the functional variant is optionally acetylated.